The brainstems of frogs contain many of the neurochemicals that are found in mammals. However, the clustering of nuclei near the ventricles makes it difficult to distinguish individual cell groups. We addressed this problem by combining immunohistochemistry with tract tracing and an analysis of cell morphology to localize neuropeptides within the brainstem of Rana pipiens. We injected a retrograde tracer, Fluoro-Gold, into the spinal cord, and, in the same frog, processed adjacent sections for immunohistochemical location of antibodies to the neuropeptides enkephalin (ENK), substance P (SP), and somatostatin (SOM). SOM+ cells were more widespread than cells containing immunoreactivity (ir) to the other substances. Most reticular nuclei in frog brainstem contained ir to at least one of these chemicals. Cells with SOM ir were found in nucleus (n.) reticularis pontis oralis, n. reticularis magnocellularis, n. reticularis paragigantocellularis, n. reticularis dorsalis, the optic tectum, n. interpeduncularis, and n. solitarius. ENK-containing cell bodies were found in n. reticularis pontis oralis, n. reticularis dorsalis, the nucleus of the solitary tract, and the tectum. The midbrain contained most of the SP+ cells. Six nonreticular nuclei (griseum centrale rhombencephali, n. isthmi, n. profundus mesencephali, n. interpeduncularis, torus semicircularis laminaris, and the tectum) contained ir to one or more of the substances but did not project to the spinal cord. The descending tract of V, and the rubrospinal, reticulospinal, and solitary tracts contained all three peptides as did the n. profundus mesencephali, n. isthmi, and specific tectal layers. Because the distribution of neurochemicals within the frog brainstem is similar to that of amniotes, our results emphasize the large amount of conservation of structure, biochemistry, and possibly function that has occurred in the brainstem, and especially in the phylogenetically old reticular formation. (C) 2001 Wiley-Liss, Inc.